Peptides are synthesized either on a solid support or in solution. In both approaches coupling and deprotection steps repetitively alternate and may be separated by intermittent purifications. In the solid phase approach, a sequence is assembled completely while attached to a solid support before it is eventually cleaved from said support. Removal of excess reagents and by-products takes place by filtration. Solid phase synthesis clearly has advantages: it is more or less generally applicable and easy to automate. However, it has also some serious drawbacks. For example, reactions are diffusion-controlled and are usually rather slow under the applied heterogeneous conditions: in order to avoid deletion sequences relatively large excesses of reagents are needed. In addition, all reactive side chains of the growing peptide must be protected: since no intermittent purifications take place, side reactions due to the presence of unprotected side chains may lead to impurities in the final product. The solid phase approach is difficult to scale up and it is costly in terms of reagents and materials.
The classical solution phase approach, on the other hand, is easier to scale up and is less expensive in terms of reagents and materials. Fully protected amino acids are usually not needed, since by-products resulting from side reactions of unprotected side chains may be removed by intermittent purifications. However, the solution phase approach requires sequence-specific protocols and the production of a complete sequence is very time-consuming.
Because of the drawbacks of these approaches there is a need for a process which combines the advantages of both these classical methods, in particular for large scale syntheses of peptides. A new process should be rapid, easy to scale up and generally applicable.
In solution phase synthesis, a slight excess of an activated carboxylic component is preferably used in each coupling step to ensure quantitative coupling to an amino component; thus the occurrence of deletion sequences in the final product can be avoided. It is usually assumed that the residual activated carboxylic component is destroyed and removed during the intermittent aqueous work-up. Insertion peptide sequences, however, are often encountered as impurities of the final peptide due to incomplete removal of residual (activated) carboxylic component after a coupling step, which subsequently has coupled following deprotection. In order to avoid the occurrence of said side reactions a scavenging step may be introduced directly after the coupling step to scavenge (inactivate) the residual activated carboxylic functions. Amines are usually applied as scavengers. The use of polyamines as scavengers leads to scavenged compounds which may be actively extracted into a—preferably acidic—aqueous phase, depending on their polarity [e.g. Kisfaludy, L. et al. (1974) Tetrahedron Lett. 19, 1785-1786]. This extraction is usually performed before the deprotection step to avoid loss of the growing peptide into the aqueous phase. However, this procedure has in numerous cases been found to result in incomplete intermittent purification due to the hydrophobicity of the scavenged compound: the intrinsic hydrophobicity of the amino acyl part of the carboxylic component is enhanced by the still present amino-protecting group. Aqueous extraction is thus not completely effective.
Recently, Carpino, L. A. et al. [(1999) J. Org. Chem. 64, 4324-4338] reported an improvement of the scavenging method. In addition to the use of a polyamine as a scavenger the amino-protecting group 1,1-dioxobenzo[b]thiophene-2-ylmethoxycarbonyl (Bsmoc) was applied in the process. The Bsmoc function has very high lability towards base. As a result thereof, residual activated carboxylic functions are scavenged and Bsmoc functions are removed in one and the same step using a polyamine. The use of the Bsmoc function has been described as a significant improvement for the production of (oligo)peptides using rapid continuous solution phase techniques allowing the assembly of a peptide in a single series of steps within a relatively short period of time.